Activation of NF-?B by anticancer treatments is thought to significantly contribute to tumor survival and clinical resistance. While inhibition of NF-?B is an effective way to sensitize cancer cells to ionizing radiation and chemotherapeutics, the development of clinical NF-?B inhibitors has been difficult. One major barrier in the development of such inhibitors is the pervasive role of NF-?B in mediating a variety of classic physiological processes, including innate and adaptive immunity, cell survival, and inflammation. This proposal aims to separate the classical NF-?B pathway from the DNA damage pathway, which contributes to cancer cell survival following treatment with anticancer agents. At the center of this proposal is NEMO, the NF-?B essential modulator. NEMO forms a number of protein-protein interactions required for activation of the DNA damage pathway. While NEMO is also a critical component of the classical pathway, I hypothesize that NEMO protein- protein interactions can be isolated to separate the classical and DNA damage-specific pathways. In order to test this hypothesis, two complementary approaches will be taken. In the biochemical approach, NEMO protein-protein interactions will be carefully mapped to the amino acid level to determine if specific protein- protein interactions can be isolated. In the functional approach, a genetic screen will be used to identify NEMO mutants that show a specific defect in DNA damage-induced NF-?B activation. In addition, novel peptide based inhibitors, developed using preliminary data, will be tested to determine their ability to specifically inhibit the DNA damage pathway. Together, these aims will allow separation of NEMO's role in the activation of the classical and DNA damage-specific pathways. Ultimately, this will facilitate the development of a targeted DNA damage-specific inhibitor of NF-?B activation.